Supplementary Information
Responding to the challenge of untreatable gonorrhea: AZD0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases
Gregory S. Basarab, Gunther H. Kern, John McNulty, John P. Mueller, Kenneth Lawrence, Karthick Vishwanathan, Richard A. Alm, Kevin Barvian, Peter Doig, Vincent Galullo, Humphrey Gardner, Madhusudhan Gowravaram, Michael Huband, Amy Kimzey, Marshall Morningstar, Amy Kutschke, Sushmita D. Lahiri, Manos Perros, Renu Singh, Virna J. A. Schuck, Ruben Tommasi, Grant Walkup and Joseph V. Newman
Table of Contents
Figures S1- S5 / S2-S6Tables S1-S7 / S7-S9
Synthetic methods / S10-S18
Biochemical methods / S18
Pharmacokinetic and distribution determinations / S18-S19
In vitro toxicology / S19
References / S20-S21
Figure S1. Key tertiary amino effect reaction to build the spiropyrimidinetrione architecture. The conversion of chiral ortho-morpholinyl aldehydes to chiral final products occurs via the tertiary amino effect reaction involving a sequential Knövenagel condensation, [1,5]-hydride shift and Mannich cyclization. During the course of the reaction, reversible epimerization can occur for the methyl substituent adjacent to the iminium species, and cyclization can occur on either face of the morpholine ring. The 9:1 product mixture reflects the thermodynamic distribution of diastereomers; the major diastereomer is separated from the minor and expresses biologically activity.1
Figure S2. Comparative haematology analysis in the rat with oral dosing of compound 6 (red) for 5 d, linezolid (orange) for 6 d and AZD0914 (blue) for 28 d. Exposures (AUC) on day 5 for 6 were 950 and 1700 µM*hr at 100 and 250 mg/kg, on day 7 for linezolid were 1900 µM*hr and on day 28 for AZD0914 were 1100±130, 2460±470 and 3820±650 µM*hr (±SD) for the 200, 500 and 1000 mg /kg doses, respectively. The data for 6 represents the average of 2 rats, for linezolid the average of 3 rats and for AZD0914, the average of 9 rats. (a) shows the reticulocytes count from the experiments. The higher dose of 6 and linezolid led to significant suppression. (b) shows lymphocyte counts with the higher dose of 6 leading to suppression. (c) shows neutrophil count with both doses of 6 and linezolid showing suppression. (d) shows the white blood cell count with the higher dose of 6 leading to suppression. AZD0914 fell within statistical equivalence of the vehicle control at the 2 lower doses. There was a statistical elevation in reticulocytes, lymphocytes and white blood cells at the highest dose. Error bars (standard deviations) are shown only above the bar graphs.
Figure S3. Mg2+ concentration effects on re-ligation of S. aureus topoisomerase/DNA/inhibitor cleavage complexes (a) S. aureus DNA gyrase ligation assay. Ciprofloxacin-induced cleaved DNA is ligated upon removal of Mg2+ by EDTA. EDTA addition results in free Mg2+ concentrations between 0.06 μM and 8 mM as indicated on the x-axis. No ligation is observed for cleaved DNA induced by AZD0914 over the entire range tested. (b) S. aureus Topo IV ligation assay. Ciprofloxacin-induced cleaved DNA is ligated upon removal of free Mg2+ by addition of EDTA. EDTA addition results in free magnesium concentrations between 0.06 μM and 8 mM as indicated on the x-axis. No ligation is observed for cleaved DNA induced by AZD0914 over the entire range of EDTA tested. The last lane on each gel marked ¢a¢ represents a control reaction without inhibitor.
Figure S4. Representative autoradiograms show rapid (5 minutes) distribution of radioactivity (dark areas) into vascularized tissue after a single IV infusion of 14C-AZD0914 to male Lister Hood rats. The dark shade for the liver tissue indicates a higher drug accumulation. The light area for the brain shows little CNS penetration. No significant penetration into vascularized eye and testis tissue is noted.
Figure S5. Time course for dosing AZD0914 in humans. Geometric mean plasma concentration-time profiles of AZD0914 following single oral doses of 200 mg to 4000 mg, from 0 to 72 hours post-dose in humans. Cmax and AUC0-14h increased as the dose was escalated. Variability in exposures in individual patients varied from 12% at the lowest dose to 46% at the highest dose. Error bars represent the standard deviation (SD).
Indication / Organism / # tested / MIC Range (µg/ml) / MIC90 (µg/ml)
Skin and skin structure infections / Staphylococcus aureus / 11640 / ≤ 0.008-0.5 / 0.252
Streptococcus pyogenes / 1358 / ≤ 0.015-0.5 / 0.252
Coagulase-negative Staphylococci / 1923 / 0.015-2.0 / 0.252
Streptococcus agalactiae / 441 / 0.03-0.5 / 0.252
Respiratory tract infections / Streptococcus pneumoniae / 2324 / ≤ 0.015-0.5 / 0.252
Moraxella catarrhalis / 145 / 0.015-0.5 / 0.252
Haemophilus influenzae / 352 / 0.03-2.0 / 0.52
Mycoplasma pneumoniae / 21 / 0.5-1.0 / 1.03
Sexually transmitted diseases / Neisseria gonorrhoeae / 473 / 0.004 to 0.25 / 0.1254-6
Chlamydia trachomatis / 10 / 0.06-0.5 / 0.253
Mycoplasma genitalium / 11 / 0.5-1.0 / 1.03
Ureaplasma spp. / 21 / 0.125-2 / 1.03
Table S2: In vitro ion channel function IC50 (µM)
Compound
Channel / Function/Effects / 1 / 5 / 6 / 7 / AZD0914
hERG7 / K+ rectifying current/QT interval / 232 / >100 / 237 / 91 / 316
hNav1.58 / Initiation of cardiac action potential/QRS interval / >316 / >80 / >333 / >100 / >333
hCav1.29 / Ca2+ entry into excitable cells/hypotension, decreased contractility/ PR interval / >100 / >100 / >100 / >100 / 326
Iks/Kv7.110 / Slowly activating K+ delayed rectifier/late repolarization/QT interval / >316 / >100 / >333 / >100 / >333
Ikr/Kv1.58 / Rapidly activating K+ delayed rectifier/QT interval / >100 / >100 / NT / >100 / >333
Ito/Kv4.311 / Transient outward K+ early repolarization/ QT interval / >292 / >100 / 224 / >90 / 216
Table S3 Single dose AZD0914 pharmacokinetic parameters in preclinical species
Parameters / CD-1 mouse (female) / Han-Wister rat (male) / Beagle dog (male) / Cynomologus monkey (male)
Dose: IV/PO (mg/kg) / 3 / 10 / 10 / 10 / 2.5 / 5 / 2.5 / 5
Clp (mL/min/kg. IV) / 70 / 22 / 4 / 12
Vss (L/kg, IV) / 3.6 / 1.2 / 0.8 / 1.3
T1/2 (h, IV) / 0.5 / 0.7 / 3.0 / 1.6
Cmax (µM, PO) / 1.7 / 1.7 / 7.1 / 6.5
Tmax (h, PO) / 1.6 / 1.8 / 3.1 / 1.6
T1/2 (h, PO) / 1.6 / 1.8 / 3.1 / 1.6
Ka (h-1) / 1.7 / 1.6 / 1.2 / -
%F / 46 / 34 / 71 / 58
PPB (%free)a / 22.1±0.3 / 19.1±1.1 / 18.4±0.3 / NDc
CLint (µL/min/106 cells)b / 7.0 / 10 / 3.8 / ND
ain vitro at AZD0914 concentration of 1-50 µM; bin vitro hepatocytes at AZD0914 concentration of 1 µM; cnot determined
Table S4. Total radioactivity (nmol/g) of 14C-AZD0914 in selected tissues following 15 min intravenous infusion (25 mg/kg) to male pigmented rats /
Tissue / Time after AZD0914 administration /
/ 5 minutes / 1 hour / 4 hours / 24 hours / 48 hours /
Adrenal cortex / 84 / 40 / 8.7 / NIe / NI
Adrenal medulla / 43 / 26 / 4.5 / NI / NI
Aortic wall / 53 / 58 / 13 / NI / NI
Bilea / 1200 / 1500 / OEc / NI / NI
Bone marrowb / 35 / 20 / 4.0 / NI / NI
Brain / 0.79 / 0.72 / BLQd / NI / NI
Choroid & retinal pigment epithelium / 16 / 17 / 8.1 / NI / NI
Heart blood / 27 / 18 / 4.2 / NI / NI
Liver / 320 / 190 / 69 / 0.35 / BLQ
Lung / 52 / 31 / 6.9 / NI / NI
Lymph node / 29 / 23 / 4.1 / NI / NI
Myocardium / 73 / 42 / 6.9 / NI / NI
Renal cortexb / 170 / 130 / 39 / NI / NI
Renal medulla (inner)b / 70 / 32 / 8.0 / NI / NI
Renal medulla (outer)b / 68 / 120 / 18 / NI / NI
Skeletal muscle / 29 / 20 / 4.2 / NI / NI
Skin, pigmented / 15 / 20 / 3.6 / NI / NI
Spinal cord / 1.0 / 1.0 / 0.30 / NI / NI
Testis / 2.4 / 5.7 / 2.6 / NI / NI
Urinea / 51 / 400 / 100 / NI / NI
White fat / 6.6 / 8.9 / NI / NI / NI
Limit of quantification / 0.30 / 0.33 / 0.24 / 0.32 / 0.24
alimit of quantification for this organ = 0.23 nmol/g; blimit of quantification for this organ = 0.27 nmol/g;
coverexposed; dbelow limit of quantification; enot identified for quantification
Table S5 Calculated fAUC/MIC parameters for AZD0914 versus S. aureus isolates in the neutropenic mouse thigh model. Values are represented as Mean ± Standard error of the Mean. /Isolate / Modal MIC (µg/mL) / Stasis / 1-log reduction /
MSSA ARC516 / 0.0625 / 98 ± 17 / 245 ± 81
MRSA ATCC33591 / 0.125 / 43 ± 5 / 96 ± 35
USA100 NRS382 / 0.125 / 43 ± 5 / 83 ± 23
USA300 NRS384 / 0.25 / 80 ± 5 / 132 ± 16
Table S6 Comparison of preclinical and clinical complicated skin and skin structure infections (cSSTI) PK/PD magnitudes associated with efficacy against S. aureus. Preclinical model magnitudes for Linezolid and Levofloxacin are represented as Mean ± Standard error of the Mean. /
Compound / PK/PD index / Clinical dose (mg/day) / PK/PD magnitude / PK/PD magnitude associated with preclinical model /
Measured / Literature
Linezolid / fAUC/MIC / 1200 / 70 / Stasis: 66 ± 25 / Stasis: 66 ± 6
Levofloxacin / fAUC/MIC / 500 / 28 / Stasis: 22 ± 2
1-log: 45 ± 15 / Stasis: 29 ± 4
1-log: 62 ± 15
Ceftriaxone / %T>MIC / 2000 / 21% / ND / Stasis: 25%
1-log: 40%
Table S7: AZD0914 in vivo effects and plasma exposure in rat and dog
Study / Salient Pathology Findings / Dose (mg/kg/day) / AUC (µg*h/mL) (±SD) / Cmax (µg/mL)
(±SD)
1 month rat (po) / NOAEL / 200 / 1100±130 / 110±30
Reduced activity, pilo-erection, irregular respiration
Seminal vesicle: secretory depletion, atrophy
Epididymis: cellular debris (minimal)
Spleen: increased congestion/haematopoiesis
Cecum & rectum: mucosal hyperplasia / 500 / 2460±470 / 170±30
As above & testes tubular degeneration partially reversible 3 months post dosing / 1000 / 3820±650 / 260±55
1 month dog (po) / NOAEL / 100 / 870±240 / 90±22
Testes tubular degeneration partially reversible 3 months post dosing / 200 / 2160±460 / 210±18
As above plus:
Epididymis cellular debris
Caecum/illium/rectum necrosis / 500 / 2780±1140 / 220±94
CV dog telemetry(single dose, iv) / NOEL / 50 / --- / 63
Emesis
Increase in cardiac contractility\relaxation and heart rate
Decrease in systolic & diastoloic blood pressure
Orthostatic hypertension / 100 / --- / 160
Synthetic Methods
General Considerations All of the solvents and reagents used were obtained commercially and used as such unless noted otherwise. 1H NMR spectra were recorded in DMSO-d6 solutions at 300 K using a Bruker Ultrashield 300 MHz instrument, a Bruker Ultrashield 400 MHz instrument or a Bruker Ultrashield 600 MHz instrument. 13C NMR spectra were recorded in DMSO-d6 solutions at 300 K and at 101 MHz using a Bruker Ultrashield 400 MHz instrument. 19F NMR spectra were recorded at 282 MHz at 300 K using a Bruker Ultrashield 300 MHz instrument. Chemical shifts are reported as parts per million relative to TMS (0.00) for 1H and 13C NMR and CFCl3 for 19F NMR. High-resolution mass spectra (HRMS) were obtained using a hybrid quadrupole time-of-flight mass spectrometer (microTOFq II, Bruker Daltonics) in ESI+ mode. Silica gel chromatographies were performed on an ISCO Combiflash Companion Instruments using ISCO RediSep Flash Cartridges (particle size: 35-70 microns) or Silicycle SiliaSep Flash Cartridges (particle size: 40-63 microns. All final compounds were determined to be greater than 95% pure via analysis by reversed phase UPLC-MS (retention times, RT, in minutes) with a Waters Acquity UPLC instrument with DAD and ELSD and a UPLC HSS T3, 2.1 x 30 mm, 1.8 um column and a gradient of 2 to 98% acetonitrile in water with 0.1% formic acid over 2.0 minutes at 1 mL/min. Injection volume was 1 µL and the column temperature was 30 °C. Detection was based on electrospray ionization (ESI) in positive and negative polarity using Waters ZQ mass spectrometer (Milford, MA, USA), diode-array UV detector from 210 to 400 nm, and evaporative light scattering detector (Sedex 75, Sedere, Alfortville Cedex, France). Optical rotations were obtained on a Jasco P-2000 instrument using a Na light source at 589 nM and a Dichrom polarizer. When not indicated, compound intermediates and reagents were purchased from chemical supply houses.
(4R)-3-{6-[(2R,6R)-2,6-Dimethylmorpholin-4-yl]-5-(1,3-dioxolan-2-yl)-7-fluoro-1,2-benzoxazol-3-yl}-4-methyl-1,3-oxazolidin-2-one A solution of 11.9 g (118 mmol) of (S)-4-methyloxazolidin-2-one12 in 20 mL DMF was added over 10 min to a stirred suspension of NaH (60% oil dispersion, 4.71 g, 117 mmol) in 140 mL DMF at 0 °C. The mixture was stirred at rt for 90 min, and a solution of (42.5 g, 117 mmol) of 3-chloro-6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-dioxolan-2-yl)-7-fluorobenzo[d]isoxazole13 in 30 mL DMF was added. This mixture was heated at 90 °C for 4 h, cooled to rt and quenched with saturated aqueous NH4Cl. Solvents were removed and the residue was partitioned between water and EtOAc. The organic layer was separated, washed with brine, dried (Na2SO4) and concentrated. The residue was chromatographed on silica gel (20-40% EtOAc gradient in CHCl3) to afford 17.5 g starting material and 14.6 g (29%) of the title compound: 1H NMR (300 MHz, DMSO-d6) d 8.23 (s, 1H), 6.18 (s, 1H), 4.66-4.84 (m, 2H), 4.18-4.30 (m, 1H), 3.91-4.14 (m, 6H), 3.22 (d, J=10.74 Hz, 2H), 2.89 (dd, J=5.27, 10.74 Hz, 2H), 1.43 (d, J=6.03 Hz, 3H), 1.20-1.30 (m, 6H); 19F NMR (282 MHz, DMSO-d6) δ -145.43 : MS (ES) MH+: 422.3 for C20H24FN3O6.